The role of neutralizing antibodies in controlling or preventing infection in SIV-infected macaques or HIV-infected humans is unresolved. The purpose of the following studies has been to more clearly define the role of antibody, utilizing the SIV/macaque model of AIDS. This work has two subprojects: the first involving purification of SIV-specific antibody from SIV- infected macaques and exploration of its efficacy when administered passively to naive macaques. The second portion of these studies has been the derivation of combinatorial macaque antibodies from SIV-infected long-term nonprogressor macaques. A trial testing the efficacy of SIV-specific immunoglobulin in modulating SIV infection of macaques was initiated in collaboration with Dr. Nancy Haigwood (Bristol Myers Squibb) and Dr. Philip Johnson (Ohio State Univ.). Immunoglobulin was purified from pooled plasma collected by plasmapheresis from a healthy SIV-infected nonprogressor rhesus macaque (E544). Initially SIVIG was tested for efficacy in modifying SIV infection when administered 24 hours after SIV challenge. High levels of SIV-specific antibodies were achieved in the recipients. Although all the macaques became infected, four of the six animals that received SIVIG exhibited a transient primary viremia followed by down regulation of virus replication such that plasma viral RNA levels were consistently less than 1000 copies/ml (the quantitation limit of the QC-PCR assay). Three animals have maintained normal CD4 numbers and are healthy at 4 years. In contrast, nine of the ten animals that received normal simian IgG or no IgG, had significantly higher virus load than SIVIG-treated animals. Eight of these macaques were euthanized due to wasting and opportunistic infections by one year post challenge. One of the surviving macaques has evidence of disease progression (i.e., CD4 decline, weight loss or diarrhea), whereas the other remains clinically healthy. This study suggests that early administration of SIVIG can have a beneficial effect in modifying subsequent disease progression. Because of the beneficial effects of polyclonal SIV immunoglobulin, we initiated a program to clone macaque SIV- specific antibodies to derive standardized reagents that could be analyzed for neutralizing activity in vitro as well as for therapeutic or prophylactic effects in vivo. For this purpose, a combinatorial phage display library of the antibody repertoire of a long term (7 years) nonprogressor SIV-infected macaque (E544) was constructed from lymph node RNA. Seven unique SIV gp120-specific Fabs were identified using recombinant monomeric SIVsmH4 gp120 produced in CHO cells (N. Haigwood), baculovirus- expressed SIVmac251 gp120, or vaccinia-expressed HIV-2 gp120. Based upon sequence and functional analyses, these clones segregate into two groups. Group 1 Fabs are cross reactive with SIVmac, SIVsm, and HIV-2 gp120, whereas Group 2 Fabs react only with SIVsm and SIVmac gp120. Two of four Group 2 Fabs neutralized SIVsm virus strains (SIVsmH4 and SIVsmB670) at nanogram concentrations, whereas they were incapable of neutralizing SIVmac251 or the neutralization-resistant SIVsmE543- 3 cloned virus. Based upon competition ELISA with mouse monoclonal antibodies which were mapped previously by Dr. Karen Kent, it appears that Group 2 Fabs bind to a conformational epitope within the V4 to V5 region of gp120, and Group 1 antibodies bind to the V3 analog of SIV gp120. The apparent narrow-specificity of the neutralizing Fabs obtained from RhE544 appears to be representative of the SIVIG purified from sequential plasma samples of this animal. The SIVIG which was used in passive immunotherapy (see above) was considerably more active in neutralizing the homologous SIVsmE660 (effective neutralization at 1.8 ug/ml) than the heterologous SIVmac251 (40 ug/ml). A two vector mammalian expression vector system for the human heavy and light chains was obtained from SmithKline Beecham. In order to produce full length macaque monoclonal antibodies, macaque heavy chain genes were amplified by PCR from bone marrow mRNA of a normal rhesus macaque to yield the macaque homologs of IgG1, IgG2, and IgG4. The macaque IgG1 gene was substituted for the human gene in the heavy chain expression vector and the VH portion replaced with that of the SIV-specific Fab. The kappa chains of the representative SIV-specific Fabs were inserted into the light chain expression vector. COS cells transiently cotransfected with the resulting macaque heavy and light chain clones secreted SIV gp120-specific antibody into the culture supernatant as demonstrated by capture ELISA. Stable antibody- expressing CHO cell lines have been selected with G418 and the level of expression is presently being optimized by exposure to sequential increasing concentrations of methotrexate. The resulting recombinant monoclonal antibodies will be evaluated for their ability to neutralize SIV in vitro as well as their ability to prevent or modify of SIV-infection when administered passively prior to SIV challenge.